DOI: 10.1002/cphc.200800434 Photoactive Branched and Linear Surface Architectures for Functional and Patterned Immobilization of Proteins and Cells onto Surfaces: A Comparative Study Petra Stegmaier [a] and Arµnzazu del Campo* [a, b] Introduction Different techniques have been developed to pattern surfaces with multiple and independent bioactive sites. Local delivery of the biological species from a reservoir can be performed by spotting, ink-jetting, or dip-pen deposition. [1, 2] Alternatively, the biological material can be patterned by contact printing using loaded polydimethylsiloxane stamps, [3–6] or adsorbed onto surfaces patterned by photolithography or self-assembly techniques after proper biofunctionalization. [7] Direct immobili- zation of the biological material to desired places on a sub- strate from a solution can also be mediated by surface layers containing complementary ligands protected with a photo- cleavable group, which can be site-selectively activated by light irradiation through a mask to build a pattern that can be recognized by the target. [8–19] Surface patterning of biomolecular species requires the pho- toactive sites to be biocompatible to prevent non-specific ad- sorption and loss of function of the arrayed material, and to reach maximal selectivity and sensitivity of the test. In the case of proteins, surfaces containing ethylene glycol (EG) units in the surface layer are well known to present antifouling proper- ties. [20–22] It has been demonstrated that the length and pack- ing density of the EG chains seem to strongly determine the protein repellence performance. A good number of studies, mostly performed with self-assembled monolayers (SAMs) of EG-containing thiols on gold substrates, have quantified the extent and relevance of these parameters. [23–26] More recently, the benefit of using branched molecular architectures in the prevention of non-specific protein adsorption has been dem- onstrated on cross-linked star-shaped polyethylene glycol (PEG) layers. [27] Although an improvement of protein repellence on such coatings could be demonstrated, [28] these films cannot directly be compared to classical grafting-to techniques in which the molecules are bound to the surface from solution without further cross-linking. Thus, a clear quantification of the impact of the branched structure has not yet been provided. This is due to the fact that dense layers of branched molecules with controlled density of functional groups and defined lengths of the arms are difficult to prepare and require long synthetic routes. This fact hinders meaningful comparison of final protein repellence performance with surface layers of analogous linear structures. Herein, we report the synthesis of organosilanes with a well- defined branched EG structure (Y-EG-NVoc (1) and Y-alkyl-NVoc (2); NVoc = nitroveratryloxycarbonyl), capable of patterning and coupling proteins to surfaces while maintaining their bio- functional state. Their molecular structure exhibits five different domains: 1) an alkoxysilane group for covalent attachment to silica surfaces, 2) a functional group capable of reacting with proteins and capped by 3) a photoremovable protecting group as built-in patterning tool, 4) a protein-repellent arm and 5) a trifunctional linker connecting the three domains through flexi- ble oligoethylene glycol (OEG) or alkyl spacers. Two different variants of this structure were synthesized, one containing only EG spacers (Y-EG-NVoc) and one containing one alkyl spacer connected to the anchoring groups (Y-alkyl-NVoc). The alkyl spacer is expected to help in the formation of more Novel photosensitive silanes with a branched molecular architec- ture combining three end-functionalized oligoethylene glycol (OEG) and alkyl arms are presented. These molecules are synthe- sized and applied to the modification of silica surfaces. The re- sulting layers are tested in their ability for the selective, patterned and functional immobilization of proteins and cells. The results demonstrate and accurately quantify the benefits of branched OEG structures against linear analogues for preventing non-spe- cific interactions with the biological material. Linear structures guarantee high selectivity for the attachment of proteins, howev- er, they fail in the case of cells. Branched structures provide good antifouling properties in both cases and allow the formation of protein patterns with higher densities of the target protein, as well as cell patterns. The results demonstrate the careful balance between surface functionality, composition and architecture that is required for maximizing the performance of any surface-based assay in biology. [a] Dr. P. Stegmaier, Dr. A. del Campo Max-Planck-Institut für Metallforschung Heisenbergstrasse 3, 70569 Stuttgart (Germany) Fax: (+ 49) 711-6893412 E-mail : delcampo@mf.mpg.de [b] Dr. A. del Campo INM-Leibniz Institut für Neue Materialien Campus D2 2, 66123 Saarbrücken (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.200800434. ChemPhysChem 2009, 10, 357 – 369  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 357